SE446153B - FAT EMULSION FOR INTRAVENOS INJECTION - Google Patents

Description

15 20 25 30 351 79os929-s 2 but it has not been clear which component of the soybean phospholipid was responsible for this. Studies of the soybean phospholipid have now shown that the cause of this side effect was in the glycolipid contained in the phospholipid. By eliminating this component, a soybean phospholipid-based emulsifier can be obtained which does not induce anemia.

Biological testing of a fat emulsion for intravenous injection must examine the amount of residual lipid in the blood as well as determine the presence or absence of such side effects as anemia after prolonged administration. Furthermore, the fat emulsion is not a natural component of a living being, but a kind of foreign substance, and it cannot be said that it has been used in vivo only because there is no residual lipid in the blood. In other words, it is desirable that injected lipid does not accumulate and remains after it has been incorporated into the reticuloendothelial system of the tissues.

In view of this, it has been considered that the accumulation and retention of lipid in the spleen and liver, which are representative of the reticuloendothelial systems of the tissues, should be considered as a basis for assessing a fat emulsion, and this has been done in connection with the present invention.

For the preparation of the emulsion according to the invention, the following three-step process can be applied. (1) Collection of a fraction containing phosphatidylcholine. * (2) Paffial hyarration (production of phospholipid with specific iodine value). (3) Separation of glycolipid.

You can get essentially the same product by performing these steps in any order, but with respect to exchange and afokfivifaf, it is best to first perform step 1 and then step 2 or 3 and finally step 3 or 2. 10 15 20 30 35 _ under MO 3, preferably 30 * 35%. 7908929-8 3 It is most appropriate to carry out the procedure in order 1, 2, 3.

Each purification step is explained in detail below.

The posfatidylcholine fraction is recovered in a conventional manner with ethanol or methanol, i.e. ethanol or methanol is added to commercial soybean phospholipid (edible or powdered lecithin), the mixture is stirred at room temperature, and the insoluble fraction is separated by decantation or filtration. The remaining soluble fraction is used as the phosphatidylcholine-containing fraction. By this process phosphatidylcholine with suitable properties as an emulsifier in concentrated form is recovered from the soybean phospholipid components. The amount of ethanol or methanol used in the process is more than twice the amount of phospholipid, preferably more than three times. The extraction temperature should be above room temperature, preferably 50 - GOOC, and the time for stirring and extraction is sufficient at about 20 minutes.

The resulting phosphatidylcholine fraction can be used, with or without concentration or evaporation of the ethanol or methanol solvent, in the next step.

The partial hydrogenation of the phospholinide is not very troublesome, and conventional hydrogenation methods can be used. In this case, the degree of hydrogenation of the hydrogenated phospholipid is 30 - 50, preferably 35 - H5, expressed as iodine value.

For example, the phospholipid is placed in an autoclave equipped with a vertically movable magnetic stirrer. Ethanol and a catalyst are added, and the mixture is combined with hydrogen. After completion of the reaction, the catalyst is filtered off, the solvent is evaporated off and hydrogenated phospholipid is recovered. The concentration of phospholipid in the solvent is in this case Raney product. nickel is used as a catalyst and is added in an amount of 3 to 30% by weight of the phospholipid. The hydrogen pressure should be about 10 kp / cm 2, and a temperature of H0 - BOOC is suitable. However, these values are related to the reaction time, and it is not necessary to limit themselves to other temperatures. 7908929-su. Given conditions, as long as one can obtain phospholipid with the iodine value 30 - 50.

Known methods can be used to separate the glycolipid. For example, the column method or a batch can; working method is applied. Silica or activated alumina can be used as a carrier. For example, silica is introduced into a column, hydrogenated phospholipid is added and developed successively with chloroform, acetone and methanol. Concentration of the methanol-eluted fraction gives only the phosphatidylcholine fraction by separation of glycolipid. In another process, hydrogenated phospholipid is dissolved in a mixture of chloroform and methanol (1: 1 by volume), activated alumina is added and the whole is stirred. The activated alumina is filtered off and the filtrate is evaporated, whereby phospholipid free from glycolipid can be obtained. Alumina is added in an amount of at least 3 times, preferably 5 times, the amount of phospholipid, and the mixture is stirred for about 30 minutes. In this case, a solvent volume of about 5 times the volume of phospholipid is sufficient.

The solvents used are chloroform-methanol, ethanol, methanol, ethanol-methanol, ethanol-hexane, ethanol-water or ethanol-dichloroethylene. The treatment temperature should be such that the hydrogenated phospholipid dissolves.

After separation of the glycolipid, the solution is subjected to aseptic filtration, such as filtration, phospholipid should be passed through a Millipore filter, after which it can be used as an emulsifier for the fat emulsion for intravenous injection.

The indicated glycolipid has the following properties.

Thin layer chromatography of crude hydrogenated soybean phospholipid on silica gel plate and development of the plate with the solvent system chloroform-methanol-water (volume ratio 65: 25: 4) gives the chromatogram shown in Figure 1.

In this chromatogram, GL (I) - GLCVÜ glycolipids, among which GL (I) are not associated with ancmi and GL (II), GL (III), Gh (IV) and GL (V) are shown side effect. Consequently, the glycolipid in question refers to the sum of GL (II) - GL (V).

In preparing the fat emulsion of the invention, emulsification may be carried out by any conventional method, such as ultrasonic treatment or high pressure emulsification. The fatty oil intended for use should meet the requirements for pharmaceutically used substances, but otherwise no restrictions apply.

According to the invention, the ratio of fatty oil to water is not limited, but in general the ratio is H - 20 parts by weight of water per 1 part by weight of oil.

The amount of purified phospholipid should be 0.05 ~ 5 parts, preferably 0.5 - 3 parts, per 10 parts of fatty oil, but a more suitable amount in practice is 0.75 - 1.0 part, in order for a full produced fat emulsion líl | Ir ~ d7slüll fl ndn fellwmuluíwn rrhållu. for intravenous administration has no side effects, such as anemia, which has hitherto been considered a side effect of fat emulsions, does not give rise to lipid accumulation in the spleen or the presence of residual lipid in the blood after administration, and is a clinically appropriate fat emulsion.

The invention is illustrated by some embodiments.

Example 1 A mixture of 5 kg of commercial grade lecithin (product from Ajinomoto) and 10 kg of 95% ethanol was stirred at room temperature for 1 hour, e: L'Ä111 <> l; -: 1 <.. ík1c -l konerrltr-'æraidefs, ooh -'íl., crsto-: lerx used soul ethanol extract. In a 6 L autoclave were placed 200 - 960 g of this ethanol extract, 1 - 3 l of ethanol were added together with a catalyst (5% Raney nickel), and this was brought into contact with hydrogen for 40 - 105 minutes for hydrogenation. The solvent was evaporated, hexane was added to the residue in a ratio of 100 ml per 150 g of residue, and the solution was filtered twice through a Millipore filter. The filtered hexane solution was added dropwise to 10 volumes of cold acetone with stirring. The precipitate formed was collected by filtration as purified hydrogenated soybean phospholipid. Depending on the time of the catalytic hydrogenation, five hydrogenated phospholipids with iodine numbers 19.7, 25.4, 36.3, H9 and 71 were obtained.

Example 2 A mixture of 1 kg of commercially powdered lecithin 1u 15 (7 from Central Soya) and 3 kg of 95% ethanol was stirred at 0 ° C for 1 min and then allowed to stand for 2 g at 35oC. The ethanol layer was separated and evaporated to give ethanol-extracted phospholipid. This phospholipid was hydrogenated in the same manner as in Example 1, and hydrogenated phospholipid (sample 1) with the iodine value 48, H was obtained.

Chromatography of 38 g of thus obtained hydrogenated phospholipid was performed with 510 g of silicic acid (Mallinckrodt) in a glass column (5.6 X 46 cm). The column was developed sequentially with 2.1 R chloroform, 2.4 L acetone and 9.2 L Methanol. The methanol fractions at 1.3-6.8 l of elucradc were recovered, the solvent was evaporated under reduced pressure, and the residue was dissolved in hexane. The hexane solution was added dropwise to cold acetone, the precipitate formed was recovered by filtration, and 21 g of purified hydrogenated phospholipid (Sample 2) was obtained. The phospholipid composition of the samples obtained is shown in Table I. Sample 2 did not contain glycolipid.

Table I Sample Neutral Fat PE PC * LPC Glycolipid%%%%% 1 3.9 7.0 77.5 3.0 8.7 2 3.0 95.0 -2.0 PE = phosphatidylethanolamine PC = phosphatidylcholine, LPC = lysophosphatidylcholine The following method for determining the phospholipid composition is applied according to the invention.

The sample is taken up on a thin-layer plate of silica gel (Merck), the plate is developed with chloroform-methanol-water (65: 25: 4, volume). After removal of the solvent, the plate is sprayed with a sulfuric acid solution of 2% cerium (IV) sulphate, and then heated at 110 ° C for 20-30 minutes, the spots of phospholipid and glycolipid turning black, so that the the chromatogram is formed. This chromatogram was scanned with a chromatogram scanner (Shimadzu model .¿ CS-910) for quantitative determination of each spot. Each (71 10 15 20 25 30 35 7908929-8 7 spot area was expressed as a percentage of the total for calculation of the phospholipid composition.

By "glycolipid content" according to the invention is meant the sum of GL (II) + GL (III) + GL (IV) + GL (V) according to Figure 1, and this is determined in the manner indicated above.

In Figure 1, the symbols have the following meanings.

GL (I) ~ GL (V) Glycolipid PL Unidentified phospholipid LPC Lysophosphatidylcholine X ~ Unidentified substance Y _ Unidentified substance TG Neutral lipid PS _ Phosphatidylserine PC Phosphatidylcholine PE Posphatidylethanolamine Example 3 In a 6 g sample ethanol in the same manner as in Example 9, 3 l of ethanol and 5% Raney nickel, after which hydrogenation was carried out at a hydrogen pressure of 15.3 kp / cm under 18 min. After the residue (sample (1: 1 by volume), 2.5 kg of active alumina es was added, and the mixture, stirring the solvent, was dissolved in 500 g of 2) in a mixture of chloroform and methanol at room temperature for 1 hour. The chloroform-methanol layer decanted, the catalyst was separated by centrifugation and filtration through a filter paper, and the solution was filtered through a Millipore filter. Upon evaporation of the solvent from the filtrate, purified soybean phospholipid (sample 3) with the iodine value H¶, 6 was obtained. The posfolipid composition in Samples 1, 2 and 3 is given in Table II. The glycolipid content in sample 3 was 2.7%. The symbols used in Table II have the same meanings as above.

Table II PPOV TG YX PL PF PC PS LPC GL 1 3.1% 2.7% 3.6% 4.3% 18.7% U5.3% 0.5 2 0.7 $ 17.6 2 11, 0 0 2, U 2.5 9.4 59.1 0 0.9 11.6 3 3.6 1.6 0 2.3 0.3 87.9 0 1.9 2.7 10 15 20 25 30 5% was added to a solution of 1 kg of soybean phospholipid, extracted in the same manner as in Example 2 and dissolved in 3 l of ethanol, Raney nickel, and hydrogenation was carried out at a hydrogen pressure of 17.4 kp / cm? under 35 min. After evaporation of the solvent, five samples of 8 g of the residue (sample 1) were dissolved with an iodine value of 39.5 each in 150 ml of a mixture of chloroform and methanol (1: 1 by volume), and these solutions were added with 8 , 15, 24, 32 and 40 g of activated alumina. The mixtures were stirred at room temperature for 1 hour, filtered, and the filtrate was passed. The filtrate was evaporated to dryness, whereby samples 2, 3, M, 5 and 6 were obtained in yields of 3.0, 3.0, 5.5, 6 5 and 7.7 g. The glycolipid levels of these samples are given in Table III. alumina through a Millipore filter.

Table III Sample Alumina Glycolipid content% 1 - 19.6 2 s I 20.9 3 16 '18.9 u 24 1ß, e 5 32' 14.3 6 no 3.5 Example 5 I A mixture of 3 kg of soybean lecithin of food grade and 11 l of 95% ethanol were stirred for 1 hour, and the ethanol layer was recovered and added with 5 volumes of acetone to give 1 kg of precipitate, which was used as ethanol-extracted soybean phospholipid. To this precipitate was added 3 l of ethanol and 5% of catalyst, and the mixture was hydrogenated at a hydrogen flow of 50 kp / cm 2 under This led to a hydrogenated soybean phospholipid with an iodine value of H0.

To a solution obtained by dissolving 5 g of hydrogenated soybean phospholipid thus prepared in 50 ml of solvent under heating was added 25 g of active alumina and the mixture was stirred for 1 hour. The alumina was filtered off and the filtrate was passed through a Millipore filter and evaporated to dryness. print. The solvents used were ethanol with 5% hexane, ethanol with 5% water, 99.5 ethanol with 5% methanol and a mixture of dichloroethylene and% ethanol, ethanol containing 5% water ( volume ratio 1: 1).

The composition of the obtained purified phospholipids is shown in Table IV. ïeäšíïill Sample Solution TG YX PL PE PC LPC GL average *%%%%%%%% 1 1 1.0 3.5 o 2.3 o 91.8, 0.2 2 2 1.5 H, 3 0 3.0 0 88.1, 0.8 3 3 1.3 H, 1 0 2.8 0 90.7, 0 u v. 1.2 3.9 o 2.3 o 91.0 1.5 oss 1.1 3.6 o 5.0 o 85.2, om.

Solution Ethanol with 5% hexane Ethanol with 5% water 99% ethanol Ethanol with 5% methanol Dichloroethylene ethanol with 5% (1: 1, v / v) the same meanings as in Table II.

(Fl-FLONÜI-Å water The symbols have ï-ïërxfuyfili _.

Five kinds of purified hydrated uoja bean phospholipid with iodine numbers 19.7, 25.4, _36.3, H9 and 71 were used. Mixtures containing 2H g of phospholipid, 200 g of purified soybean oil, 50 g of glycerol JP, and 1726 g of distilled water for injection were emulsified in a high pressure emulsifier (Manton-Gaulin) under nitrogen flow at 650 kp / cm? print. The fat emulsions thus obtained were filtered through a Millipore filter (1.2 μm pores), and the filtrate was sterilized by heating at 120 ° C and 1 atm for 20 minutes, after which they were used in animal experiments.

Male Sprague-Dawley (SD) strains, weighing 150 g, were injected into the tail vein of 60 ml / kg body weight of fat emulsion continuously for 10 days, using all five emulsions.

The injection rate was 3-5 ml / min. After successive injection for 10 days, the rats were left without food overnight. Their spleens were taken out to determine neutral fat therein by a conventional method.

The results of these animal experiments are shown in Figure 2.

High hematocrit values and low levels of neutral lipid content and phospholipid content were present in animals in which fat emulsions containing hydrogenated phospholipids with iodine value in the range of 30 - 50 were injected.

In 2, E] means neutral lipid content in the spleen, phospholipid content in the plasma, and <> neutral lipid- T figure x hematocrit, o content in the plasma.

Example 7 Samples 1 and 2 of Example 2 were emulsified in a high pressure emulsifier using the same method as in Example 6, and heat sterilized to give fat emulsions for intravenous injection. The emulsification took place under a pressure of 600 kp / cm2 and was repeated 6 times.

The resulting fat emulsions were injected into the tail veins of male SD strains, weighing about 200 g, at a dose of 60 ml / kg body weight. The rats were left without food overnight, after which their spleens were removed to be examined for the accumulation of neutral fat. Control animals were injected with a fat emulsion prepared with commercial egg yolk phospholipid or physiological saline. The results are shown in Table V.

BEL! e Group Residual amount of neutral fat in spleen Ing / g '1 12.5 12.89 2 ~ 2.10 10.12 3 1H, 26 t8.07 4 2.16 t0.27 Each group: n = 3 Group 1: Emulsion with sample 1 in example 2 2. H II H Sample 2 3: Emulsion with commercial egg yolk phospholipid 4: Physiological saline - The fat emulsions obtained as above were administered into the tail vein of male SD strains, weighing about 150 g, at a dose of 100 ml / kg body weight for 10 days in sequence. The rats were then left without food overnight, after which the blood, spleen and liver were taken and the hematocrit value, erythrocyte, the value, the neutral fat content and the phospholipid content were measured. Fat emulsion prepared with commercial egg yolk phospholipid was administered as a control. The results are given in Table VI.

Tables V and VI show that the fat emulsion with soybean phospholipid, from which glycolipid has been separated after hydrogenation, is suitable for intravenous injection.

T_abe11 VI Group Hemato- Erythrocytes Lipid in Neutral fat in V chalk 104 / mm3 plasma (mg / dl) organ (mg / g)% Neutral- Phospho- Spleen Liver fat iipid 1 HU, Ûi1,1 66518 69i1U 299i27 2,99i0,28 52 i 1.01 H3.3il, 1 642162 62i7 3U6t1U1 4.63i0.62 25.3il1.8 46.3í1.1 66Hi28 40i6 129i9 2.81tO .32 14.91 3.5 Each group: n = 3 Group 1 : Emulsion with sample 2 in Example 2 2: Emulsion with commercial egg yolk phospholipid 3: Control without administration of fat emulsion Example 8 Fat emulsions were obtained using the phospholipid in sample 3 according to Example 3 and emulsified by the method of Example 6. These fat emulsions were injected into the tail veins of rat males of strain SD, weighing 140 g, at a dose of 100 ml / kg body weight for 10 consecutive days. The injection rate was 3 - 5 ml / min. Various studies were performed and compared with control rats, which were given an emulsion prepared with commercial egg yolk lecithin or physiological saline, and with untreated rats. The results are given in Table VII. 1oi 15 207 25 30 35 7908929-8 12 'Table VII Group Hematocrit Erythrocytes Lipid in plasma (mg / dl)% I 104 / mm3 Neutrallipid Phospholipid 1 u3, a: 1,3 suoils' sa: 1o 1su119 2 us, 1: o, s 669112 1o5: 11 zsufss 3 us, s: o, 9 easizvn ssi1o 1o9i12 du us, o11.2 sulis sziu losiv Each group: n = 4 Group 1: Fat emulsion with sample 3 in example 3 2 Fat emulsion with commercial egg yolk phospholipid 3 : Physiological saline H: Untreated control Example 9 Fat emulsions were obtained with phospholipid prepared according to Example 4 (Samples 2 - 6) and emulsified by ultrasonic treatment. The emulsification conditions were 25 kHz for 2 hours. The resulting fat emulsions were injected into the tail veins of male SD strains, weighing 200 g, in a single dose of 80 ml / kg body weight, and accumulated residual neutral fat in the spleen was determined. The results given in Table VIII indicate that the lower the amount of glycolipid, the lower the amount of neutral fat in the spleen.

Table VIII Residual amount of neutral fat in the spleen ms / g 169.2i15.5 55.7i25, H 29.91 7.1 21.9i13.0 5 20.91 7.8 Group -FGOIQI-Å Each group: n = 3 Group 1 : Fat emulsion with sample 2 in example 4 2: "sample 3 in" 3: "sample 4 in" H: "sample 5 in" 5: "sample 6 1" 10 15 20 25 30 7908929-8 13 Example 10 A fat emulsion was prepared using Sample 1 in Example 5 and using the same method as in Example 6.

The emulsion was administered to rats in the same manner as in Example 8. The rats weighed about 110 g. The results are given in Table IX.

Table IX Group Hematocrit Erythrocytes Plasma Lipid (mg / dl) 1o ”/ mm3 Neural Rally Lipid. 108i13 Each group: n = U Group 1: Fat emulsion with sample 1 in Example 5 2: Fat emulsion with commercial egg yolk phospholipid 3: Physiological saline Example 11 The fat emulsion used for group 1 in Example 8 was used to study acute toxicity with strains of strain SD, weighing about 200 g. The LD50 value obtained was 1H2 ml / kg body weight. Subacute toxicity testing was performed on 30 consecutive adjuvants of 20 or H0 ml / kg body weight, but no rats died. Their body weight increased at the same rate as in rats, which received physiological saline, and there were no signs of anemia. The saline solution prepared according to the invention was thus found to be completely safe.

Example 12 Purified soybean phospholipid having a glycolipid content of 0 - 11.6% was obtained by the same methods as in Examples 2, 3 and 4.

These were used as emulsifiers, and the emulsification was carried out in the same manner as in Example 7, whereby fat emulsions for intravenous injection were obtained. These emulsions were injected into the tail veins of male SD 1 rat at a dose of 100 ml / kg body weight for 10 consecutive days. The rats were left without food overnight after the end of the injections, blood was collected from the lower half, and the erythrocyte value was determined. Control rats received injections of physiological saline. The erythrocyte value in the control blood was set to 100, and the erythrocyte values in rats injected with fat emulsion were expressed in relative numbers. The relationship between the glycolipid content and the relative erythrocyte value is shown in Figure 3. This shows that anemia does not occur at a glycolipid content below 5%.

Claims (1)

7. 7308929-s _PATENTRAL 1 5 Fat emulsion for intravenous injection consisting essentially of oil, water and an emulsifier, characterized in that the emulsifier is a purified phospholipid of soybean origin, the glycolipid content of which is less than 5% and has a degree of hydration of 30 - 50 expressed by the iodine value.